Kazuo Chihara

Leptin Induces Mitogen-activated Protein Kinase- dependent Proliferation of C3H10T1/2 Cells

Leptin, secreted by adipocytes, regulates satiety and energy expenditure. Several forms of leptin receptors produced by alternative mRNA splicing are found in many tissues, including the hypothalamus, liver, lung, kidney, hematopoietic cells, and gonads, suggesting that leptin exerts effects in these tissues. In accordance with the distribution of leptin receptors, there is accumulating evidence that leptin plays various roles in reproduction, hematopoiesis, and the immune systems in addition to the regulation of food intake and energy expenditure. In the present study, we examined the in vitro effects of leptin on proliferation of a mouse embryonic cell line, C3H10T1/2, and its mechanism of action. Leptin caused a dose- and time-dependent increase in mitogen-activated protein kinase (MAPK) activity that was accompanied by an increase in C3H10T1/2 cell number. The MAPK kinase-1-specific inhibitor PD98059 completely blocked the increases in both MAPK activity and cell proliferation caused by leptin. These findings indicate that leptin stimulates the proliferation of C3H10T1/2 cells via the MAPK cascade.

Short Stature Caused by a Mutant Growth Hormone

The causes of growth hormone–dependent short stature are primary pituitary disease, pituitary deficiency due to hypothalamic dysfunction, and, less often, insensitivity to growth hormone. The prototypical syndrome of growth hormone insensitivity is Laron-type dwarfism, which is characterized by absent or defective growth hormone receptors. Kowarski et al. described two children with growth retardation resulting from biologically inactive growth hormone1; additional cases were reported subsequently.2–7 This disorder is characterized by high serum concentrations of immunoreactive growth hormone, low serum concentrations of insulin-like growth factor I (IGF-I), and increases in both serum IGF-I and linear growth after the administration of .xa0.xa0.

Biologically inactive growth hormone caused by an amino acid substitution.

Short stature caused by biologically inactive growth hormone (GH) is characterized by lack of GH action despite high immunoassayable GH levels in serum and marked catch-up growth to exogenous GH administration. We found a heterozygous single-base substitution (A-->G) in exon 4 of the GH-1 gene of a girl with short stature, clinically suspected to indicate the presence of bioinactive GH and resulting in the substitution of glycine for aspartic acid at codon 112. We confirmed the presence of mutant GH in the serum using isoelectric focusing analysis. The locus of mutation D112G was found within site 2 of the GH molecule in binding with GH receptor (GHR)/GH binding protein (GHBP). The expressed recombinant mutant GH tended to form a 1:1 instead of the 1:2 GH-GHBP complex normally produced by wild-type GH. The formation of a 1:2 GH-GHBP complex is compatible with the dimerization of GHRs by GH, a crucial step in GH signal transduction. Mutant GH was less potent than wild-type GH not only in phosphorylation of tyrosine residues in GHR, janus kinase 2 (JAK2), and signal transducers and activators of transcription 5 (STAT5) in IM-9 cells, but also in metabolic responses of BaF/GM cells, a stable clone transfected with cDNA of the chimera of the extracellular domain of human GHR, the transmembrane and the cytoplasmic domain of the human thrombopoietin receptor. These results indicate that the D112G mutation in the GH-1 gene causes production of bioinactive GH, which prevents dimerization of GHR and is therefore responsible for the patients short stature.

Growth Hormone (GH)‐Releasing Peptide and GH Releasing Hormone Stimulate GH Release from Subpopulations of Somatotrophs in Rats

The synthetic hexapeptide GH‐releasing peptide (His‐D‐Trp‐Ala‐Trp‐D‐Phe‐Lys‐NH2; GHRP‐6) and GH releasing hormone (GHRH) are both potent stimulators of GH release in rats. Using reverse hemolytic plaque assay (RHPA), we have compared the effects of human GHRH and GHRP‐6 on GH release from the dispersed individual cells of rat anterior pituitary.

CHARACTERIZATION OF GH3 CELLS OVEREXPRESSING BASIC FIBROBLAST GROWTH FACTOR (FGF-2)

Basic fibroblast growth factor (FGF‐2) is not only a potent mitogen for various cells but also a multifunctional factor with angiogenic and chemotactic activity, and the capacity to induce the synthesis of various proteinases and to modulate endocrine function. To clarify the role played by FGF‐2 in the progression of pituitary tumor, we fused rat FGF‐2 cDNA to the promoter SRα, consisting of the early promoter of SV40 and HTLV(I)‐LTR, and we cotransfected GH3 cells with pSV2‐neo by an electroporation method. After selection by G418, we obtained 7 neomycin‐resistant clones. Southern blot analysis of genomic DNA revealed the presence of transfected rat FGF‐2 cDNA in 4 of the 7 clones. To measure FGF‐2 molecules, we established a new immuno‐fluorometric assay system, using 3 monoclonal antibodies against different portions of human FGF‐2. This assay had a minimum sensitivity of 10u2003pg/ml and cross‐reacted neither with acidic fibroblast growth factor (FGF‐1) nor insulin‐like growth factor 1 (IGF‐1), even at a concentration of 100u2003ng/ml. Although FGF‐2 was undetectable in the culture medium of any of the clones, the cell homogenate contained a significant amount of FGF‐2 (7.2u2003ng/mg protein) in 1 of the 4 FGF‐2‐transfected clones (GH3FGF(+)), whereas FGF‐2 was not detected (<5.2u2003pg/mg protein) in the cell homogenates of either the parent GH3 cells or the control cells transfected with pSV2‐neo alone (GH3FGF(−)). GH3FGF(+) grew as adherent cells and formed epithelial sheets with a growth rate similar to that of control cells. The amount of prolactin(PRL) released by TRH was greater in GH3FGF(+) than that in GH3 or GH3FGF(−). On the other hand, the sensitivity to SRIF was increased in GH3FGF(+) compared with that in other clones. The findings of these in vitro studies indicate that FGF‐2, if it is expressed in pituitary tumor cells, plays little if any role in cell growth but may modulate certain cell functions such as responsiveness to hormones.